Immunologic Effect of Polysaccharides Extracted from Sipunculus Nudus (SNP) on Hepatoma Hepg2-Bearing Mice

bioRxiv preprint doi: https://doi.org/10.1101/175190; this version posted August 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Immunologic Effect of Polysaccharides Extracted from Sipunculus nudus (SNP) on Hepatoma HepG2-bearing Mice Jie Sua,b, Linlin Jiangb, Jingna Wub, Zhiyu Liub,*,1 and Yuping Wua,*,1 a South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Guangdong Provincial Key Laboratoryof Marine Resources and Coastal Engineering, Zhuhai Key Laboratory of Marine Bioresources and Environment, School of Marine Sciences, Sun Yat-SenUniversity, Guangzhou 519000, PR China b Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province, Fujian Collaborative Innovation Center for Exploitationand Utilization of Marine Biological Resources, Fisheries Research Institute of Fujian, Xiamen, Fujian 361012, PR China * Authors for corresponding : email addresses: [email protected] (Z. Liu), [email protected] (Y. Wu). 1 These authors contributed equally. bioRxiv preprint doi: https://doi.org/10.1101/175190; this version posted August 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Summary statement SNP(polysaccharides extracted from Sipunculus nudus) mediates anti-tumor activity through influencing immunoregulation, and SNP can be explored as a promising candidate for future anticancer drug. Abstract Since many studies have clarified the biological activity of polysaccharides, we investigated the effect of SNP which was the water-soluble polysaccharides extracted from Sipunculus nudus on Hepatoma HepG2-bearing Mice to verify the potential of SNP as an effective clinical agent for liver cancer therapy. SNP were administered at the doses of 50,100, and 200 mg/kg to HepG2-bearing mice to determine their antitumor effects. SNP had an inhibitory effect on the growth of HepG2 cells and enhanced the immunological effect on HepG2 tumor-bearing mice. SNP increased the expression of IL-2, IFN- γ, and TNF-α cytokines in serum, suggesting that SNP can strengthen the antitumor immune response. In addition, SNP increased ATF4, DDIT3, and IkBα expression and decreased CYR61, HSP90, and VEGF expression, all of which are proteins involved in antitumor activity and cell death/ survival. our results suggested that SNP may be a novel antitumor agent. Keywords: Polysaccharides; Sipunculus nudus; Hepatoma; Cytokines; Introduction Hepatitis B (HBV)-associated hepatoma is one of the leading causes of cancer death in Asia(Sun et al, 2011). Currently, the treatment of hepatoma includes surgery and intervention chemotherapy; however, these measures often have a variety of side effects (Paraskevi et al, 2006). Therefore, identification of non-invasive treatments is in great interest for hepatoma therapy that may increase the survival quality of cancer patients. SNP, the main ingredient in Sipunculus nudus extract, is a monosaccharide that mainly comprises L-rhamnose, L- arabinose, D-ribose, D-glucose and D-galactose(Liu et al, 2011). In vivo and in vitro studies have indicated that SNP intake may enhance immune function (Cui et al, 2014). We previously demonstrated the pro-apoptotic activity and anti-HBV virus activity of SNP on HepG2.2.15 cells (Su et al, 2016). In the present study, we show that SNP can stimulate immunological function to mediate antitumor activity. Further development and exploration of SNP may identify SNP as an effective antitumor agent for clinical liver cancer therapy. Materials and methods Isolation and purification. Referring to previous reports (Su et al, 2016), S.nudus (diameter 6 ± 2 mm, length 10 ± 2 cm) was collected from Xiamen market, China. Fresh peanut worms were washed, oven dried, and crushed into powder. The powder was hydrolyzed in trypsin at 50 °C for 5 hours, and the insoluble material was removed by centrifugation. The supernatant liquor was deproteinized 4 times using the Sevage method（Staub，1965； aZhang et al, 2011）. Next, 4 volumes of cold ethanol was added to precipitate the material after standing at 4 °C overnight (b Zhang CX et al, 2011). The resulting precipitate was then centrifuged (3000 g × 10 min). After washing with ethanol 3 times, the precipitate was freeze-dried in vacuo and ground into a powder to produce a crude product. The crude product was subjected to a DEAE-Sepharose anion exchange column (3.0 cm × 40 cm), eluting at 0.5 ml/min successively with 0.0175 M pH 6.7 phosphate buffer solutions. Each fraction was collected with 2 ml of elute. The main elution fraction containing the carbohydrates was concentrated, dialyzed, and bioRxiv preprint doi: https://doi.org/10.1101/175190; this version posted August 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. lyophilized. Mice assay.Male BALB/c mice (6-8 weeks old; 18～22g) were obtained from Shanghai Slack Laboratory Animal Co. Ltd. All mice were housed under conditions of 20-25 room temperature, 30%-70% relative humidity, and 12-hour light/ dark cycle. Animals were fed with the standard mice food and provided tap water ad libitum. Cell culture. The HepG2 cell line was obtained from ShangHai MeiXuan Biological Technology Co. Ltd. and cultured in DMEM medium with 10% fetal bovine serum (FBS). Cells were incubated at 37 in 5% CO2 humidified air. Experimental models.A total of 100 mice were used in this study and divided into two parts: treatment and prevention. For the treatment group, 50 mice received a subcutaneous inoculation of HepG2 cells (2×106 cells per mouse). A week after tumor cell inoculation, we screened 30 mice for millet size tumors and used as the experimental animals. The experimental animals were randomly divided into five groups of 6 mice each----vehicle control (saline), astragalus polysaccharides (APS; 100 mg/kg), and three SNP groups (50,100, and 200 mg/kg). For the prevention study, the other 50 mice were randomly divided into four groups--- preconditioning vehicle and three SNP groups (50,100, and 200 mg/kg). The mice were initially administered drugs according to respective treatment groups by intragastric administration for 1 month. Next, the animals received a subcutaneous inoculation of HepG2 cells (2×106 cells per mouse). A week after injection, 30 mice were screened for millet size tumors, divided into 6 mice/group and used as the experimental models. Antitumor assays. Drugs were given per group once daily intragastrically for 16 consecutive days after tumor screening. The vehicle group consisted of normal saline intake mice (0.2 ml each mice). All animals were humanely killed and tumors, thymuses, and spleens were dissected and weighed at the end of the assay. Body weights, tumor weights, thymuses weights and spleens weights were recorded. The tumor inhibition rate and organ index were calculated using the following equation: Tumor inhibition rate = (mean tumor weight of vehicle group – mean tumor weight of treated group)/mean tumor weight of vehicle group×100% Organ Index=organ weight/ body weight×100% Histology. At the end of the antitumor assay, tumors of all mice were collected for histological examination. These tissues were fixed in 4% paraformaldehyde solution, embedded in paraffin, and serially cut for hematoxylin and eosin (H&E) staining. Samples were imaged under a light microscope for morphological observation. Cytokines assay. At the end of the antitumor assay, blood samples were obtained from the sinus by extracting the eyes of the mice. After 2h’ incubation, blood serum was separated by centrifugation at 3000 rpm, 10min. Cytokines including interleukin (IL) 2, interferon (IFN) γ, and tumor necrosis factor (TNF) α were determined by enzyme linked immunosorbent assay (ELISA). Western Bloting. In the Western blot analysis, 50mg of each tumor was harvested, and these samples were added in cell lysis buffer. After trituration and centrifugation, the concentration of protein per group was determined using a BCA protein assay kit according to the manufacturer’s protocol. Then 20μg of total protein was used for SDS-PAGE, proteins were transferred to membranes, and blocked with 5% skim milk. ATF4, DDIT3, IkBa, CYR61, HSP90 and VEGF primary antibodies were added separately and the sections were incubated at 4℃ overnight. Subsequently, membranes were washed in PBS and secondary antibodies were added and incubated bioRxiv preprint doi: https://doi.org/10.1101/175190; this version posted August 11, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. correspondingly. After washing with PBS, the membranes were stained with ECL, and observed on an automatic electrophoresis gel imaging analyzer. Protein levels of above mentioned interest protein were analyzed by comparing with an internal reference. Statistical analysis. Data was expressed as means ± standard deviation (S.D.). Student’s t test was used to analyze the differences between the control and test groups. Differences were considered to be statistically significant at P < 0.05 and very significant at P < 0.01. Results Isolation and Purification. Based on previous reports, polysaccharides were obtained after fractionation on a DEAE-Sepharose anion exchange column from S.nudus. SNP comprised Ara 10.7%, Rha 12.6%, Gal 16.4%, Glu 31.3%, Xyl 18.2%, and Man 10.8 %( Su et al, 2016).

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